SUMMARY The rate of prematurity in the US is currently 1 in 10 births, resulting in large numbers of infants that are vulnerable to both short-term and long-term health problems. Preterm Premature Rupture of Membranes (pPROM) causes thirty percent of preterm birth, with almost half of these cases due to infection. Although the infectious agent can often be identified, little can be done to halt its progression or identify those who are at risk. The central premise of this work is that understanding the normal mechanisms of membrane rupture will help to understand how they weaken in normal term pregnancies and how this may be altered in pPROM. This proposal tests the hypothesis that Danger Associated Molecular Pattern (DAMP) ligands for the Toll-like receptors (TLR) have a key role in the initiation of inflammation and the resultant weakening of the amnion in normal rupture of the membranes. This drives the predictions that (1) DAMPs are produced in the amnion in response to cell stress, and also (2) that DAMPs and the TLR infection ligands, Pathogen Associated Molecular Patterns (PAMPs), activate TLR on the mesenchymal cells (AMC) of the amnion causing its weakening and rupture through different inflammatory signatures. Specific Aim 1: Ascertain if and how DAMPs are produced in mesenchymal amnion cells. Hypothesis: The amnion produces DAMP signals that act locally to initiate fetal membrane weakening. Approach: Normal term human amnion samples will be utilized to detect the production of various TLR activating DAMPs by immunocytochemistry, western blotting and ELISA. Human AMC will be grown in culture and subjected to stretch or oxidative stress and the resultant DAMP production measured. Outcomes: The data will link the known term causes of cell stress with the DAMP/TLR pathway, supporting them as the key initiators of sterile inflammation that results in rupture at term. Specific Aim 2: Determine the mechanism(s) by which DAMPs weaken the human amnion via TLRs and compare that to the response seen to PAMPs. Hypothesis: TLR on AMC are activated by DAMPs and PAMPs to drive extracellular matrix degradation and apoptosis through the initiation of different inflammatory signatures. Approach: AMC will be cultured in 3D AlvetexTM to measure the effect of known DAMPs on the enzymatic control of the extracellular matrix, cell viability and inflammation. The inflammatory signature for TLR2/6 after activation by DAMPs and PAMPs will be compared by cytokine secretion and the temporal analysis of miRNA expression. Outcome: This data will establish the mechanisms of TLR amnion weakening by sterile inflammation and help to understand why the clinical outcomes seen during normal membrane rupture and infection driven rupture are different.